Platelets are one of the primary components of human blood. Blood is basically made up of plasma, red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Platelets are produced in the bone marrow by large cells called megakaryocytes. It is commonly understood that platelets are actually not true cells, but are fragments of membrane and cytoplasm containing granules. More specifically, platelets comprise an outer membrane and cytoplasm from megakaryocytes which in turn contain granules, dense bodies, a dense tubular system, and mitochondria.
It is well recognized that platelets are an essential component of the blood clotting process and play a vital role in controlling bleeding. They adhere specifically to the endothelial cells and the basement membrane lining of damaged blood vessels, where they trigger and participate in hemostasis or clotting. In addition, inflammatory mediators may be released in response to this contact or in response to the mediators released by damaged tissue or other platelets. Important mediators released by platelets include serotonin and coagulation factors. Damaged blood vessels or other vascular breaches are repaired by platelets through such adhesion, and the ensuing response to this type of damage is further amplified by platelet secretions resulting in platelet aggregation and fibrin formation or a stabilized clot.
Platelet transfusions are an important aspect of the clinical management of patients with low numbers of platelets. Normal platelet counts range from about 150,000 to about 400,000 per cu/ml. A relatively low number of platelets may be due to cancer treatment and other reasons. Some patients may require transfusions for hemostasis, or whose platelets are defective in function. Platelets normally aggregate at a site of injury or vessel breakage as described above, and release a number of mediators to which other platelets respond in an amplifying biologic effect or coagulation cascade, which in turn stimulates other biologic effects. The normal, circulating platelet has a disc-shaped morphology. In response to a stimulus, the discs swell into spheres, and may further swell to a point where they eventually rupture. Concurrent with this observed change in shape, platelets release a variety of mediators, many of which are released by granules contained within the platelet. The morphology of platelets can be generally determined by microscopic observation. The ability of platelets to maintain their morphology can be tested by subjecting them to mild hypotonic conditions and following their return to disc shape as the membranes pump out excess water. This test is called hypotonic shock response (HSR) and ascertains the ability of the platelet membrane to remain intact during swelling of the platelet and to function by pumping water out of the platelet. Another test of platelet function monitors the change in platelet shape as platelets swell in response to a stimulus. This test is called extent of shape change (ESC). These methods are well known, and there is commercial instrumentation for determining these measures.
The process of preparing platelet transfusions typically begins with the separation of platelets as a product from whole blood. Bags of concentrated platelets in blood plasma may be obtained by apheresis or pheresis (centrifugal separation during the donor process while other components are returned to the donor) or by selective removal from whole blood after gravity or centrifugal sedimentation of blood cells. Preparation by centrifugation of whole blood collected in anticoagulant can be either with a slow spin that leaves platelets in suspension while removing red cells, followed by a faster spin to sediment the platelets from plasma, allowing resuspension in a reduced volume of plasma (slow/fast method producing platelet concentrate), or with a fast spin that sediments red cells and platelets, the platelets being in a buffy coat on top of the red cells, followed by removing that buffy coat layer along with an amount of plasma and doing a slow spin to remove remaining red cells from the suspended buffy coat (fast/slow method producing buffy coat platelets). Typically, sodium citrate is the anticoagulant used in making platelet preparations and the final concentration of citrate is up to about 15 mM in the platelet product in plasma.
It is very important to preserve platelets after their isolation from the body under suitable conditions that not only maintain the biological activity of the platelets, but also keep them suitable for subsequent clinical use. The average survival time for a platelet in the body after it leaves the bone marrow is eight to ten days. The average expected survival time for circulating platelets is four to five days, which is the average for an entire platelet population. Meanwhile, the current standard and approved method for platelet storage is in a platelet bag that is stored at room temperature for not more than five (5) days. This storage time is limited by the effects of metabolism, including changes in pH, the loss of clinical usefulness, and the risks from growth of small numbers of bacteria that may contaminate the preparation. Some clinicians apply even stricter criteria and decline to use platelets stored for more than three (3) days. The relatively short storage times and the risk of bacterial growth during such storage are major disadvantages and problems associated with current platelet storage methods.
Today, some platelets in suspension are also stored at reduced temperatures within normal refrigeration or freezing temperatures ranges. While cold temperature generally serves to suppress bacterial growth, platelets at refrigerator temperatures are known to become activated, change shape, lose function, and are cleared from the circulation if transfused. Thus, cold storage has been deemed to render platelets non-functional and of little clinical use. Other approaches for preserving platelets have also been reported, including cryopreservation at freezing temperatures in the presence of cryoprotectant such as DMSO. This freezing process is tedious, typically involving gradual lowering of temperature. The recovery of platelets from cryopreservation is also tedious and requires the removal of DMSO and/or other components prior to use in transfusion. Expected platelet recovery from the effects of freezing itself can be relatively low, and the yield is further reduced by subsequent washing in order to remove cryoprotectants or other agents. Satisfactory clinical use has not yet been reported for such platelet preservation techniques.
The present invention provides solutions and methods for cooling blood platelets to refrigerator temperatures and for storage of platelets at refrigerator temperatures for many days. The platelets stored using these methods remain functional and clinically useful.